CN110331121A - A kind of recombinant bacterium of high yield lipopeptid and its application - Google Patents

Abstract

The invention discloses a kind of recombinant bacterium for the high yield lipopeptid for belonging to gene engineering technology field and its application, the recombinant bacterium of the high yield lipopeptid is that the 2-Isopropylmalate synthase genetic transformation in leucine route of synthesis is built-up to original strain.The recombinant bacterium of further overexpression part leucine synthesis path (2-Isopropylmalate synthase, 3-Isopropylmalate dehydrogenase, 3-Isopropylmalate dehydratase and branched-chain amino acid transaminase) of the invention is compared with starting strain, Surfactin yield highest improves 55.6%, it can be used for the production of Lipopeptide Biosurfactants, with good prospects for commercial application, lipopeptid yield average out to 13-16g/L in fermentation liquid obtained by shake flask fermentation.

Description

A kind of recombinant bacteria with high lipopeptide production and its application

technical field

The invention belongs to the technical field of genetic engineering, and in particular relates to a high-production lipopeptide recombinant bacterium and its application.

Background technique

Lipopeptide biosurfactants are a kind of amphoteric substances connected by hydrophilic cyclic oligopeptides and hydrophobic fatty acid chains with lactone bonds, and are mainly synthesized by microorganisms such as Bacillus and Streptomyces. Due to the difference in amino acid composition and ring formation in the peptide rings that make up lipopeptides, lipopeptides from Bacillus can be classified into surfactin, phenmusin, iturin, etc. Among them, the unique "saddle" conformation is formed at the air/liquid interface. Surfactin has good surface activity, biodegradability and antibacterial activity, and has broad application prospects in the fields of oil exploration, biological control, medicine and daily chemicals. However, the low yield of surfactin produced by Bacillus fermentation limits the industrial production and application of surfactin.

At present, methods to improve the fermentation level of microbial lipopeptides include optimization of culture conditions, mutation breeding, enhancement of surfactin transmembrane transport, enhancement of surfactin synthase expression, and enhancement of fatty acid precursor supply, etc. Patent documents (CN101892176A, CN 101775427A, WO 2002026961A) etc. have improved the yield of surfactin in the fermentation broth by optimizing the culture medium and culture conditions during the fermentation process of Bacillus subtilis to produce surfactin. In terms of mutation breeding, CN101928677A discloses the treatment of Streptomyces roseospora by ultraviolet mutagenesis, and US05227294 discloses the treatment of Bacillus subtilis with nitrosomethylurethane. volume has increased. In terms of genetic engineering, the Chinese patent document CN103898038A discloses that by strengthening the transmembrane protein YcxA that transports lipopeptide from intracellular to extracellular, the yield of surfactin produced by Bacillus subtilis fermentation is increased by 97%. Chinese patent document CN1554747A discloses that the expression of comA gene in Bacillus subtilis increases the lipopeptide production by 50%. In addition, fatty acids and amino acids in Bacillus subtilis catalyze the synthesis of surfactin under the action of surfactin synthase. Chinese patent document CN105400784A replaces the lipopeptide synthase gene cluster promoter with an inducible strong promoter Pg3, and surfactin Yield increased by 17.7 times. Chinese patent document CN109097315A increases the yield of surfactin by 47% by overexpressing biotin carboxylase YngH in the branched-chain fatty acid synthesis pathway. (Qun Wu, Yan Zhi, Yan Xu, Systematically engineering the biosynthesis of a green biosurfactant surfactin by Bacillus subtilis 168 [J]. Metabolic Engineering, 2019, 52:87-97) disclosed that strengthening the whole pathway of intracellular branched-chain fatty acid synthesis improves The supply of fatty acids in the body, thereby increasing the production of surfactant by 20.8 times. Another major precursor amino acid for surfactin synthesis, (Coutte F, Niehren J, Dhali D, et al. Modelingleucine's metabolic pathway and knockout prediction improving the production of surfactin, a biosurfactant from Bacillus subtilis[J].Biotechnology Journal, 2015,10(8SI):1216-1234) disclosed that the addition of leucine in the medium can increase the yield of surfactin by 3 times, indicating that increasing the content of leucine is an effective strategy to increase the yield of surfactin. However, (Qun Wu, Yan Zhi, Yan Xu, Systematically engineering the biosynthesis of a green biosurfactantsurfactin by Bacillus subtilis 168[J]. Metabolic Engineering, 2019, 52:87-97) overexpressed in surfactin-producing host bacteria Acetolactate synthase AlsS, ketol acid reductoisomerase IlvC, dihydroxyacid dehydratase IlvD, 2-isopropylmalate synthase LeuA, 3-isopropylmalate dehydrogenase in the leucine synthesis pathway Enzymes LeuB and 3-isopropylmalate dehydratase LeuCD, Surfactin production did not increase. There is no report on the research on enhancing the synthesis of leucine in cells to effectively increase the production of surfactin.

Contents of the invention

In order to overcome the shortcomings in the prior art, the object of the present invention is to provide a highly efficient lipopeptide-producing recombinant bacterium, which can increase the lipopeptide production.

The recombinant bacterium is constructed by transforming the 2-isopropylmalate synthase gene in the leucine synthesis pathway into the original strain.

In the above-mentioned recombinant bacteria, optionally, the recombinant bacteria are also transferred to one or one of the 3-isopropylmalate dehydrogenase gene, the 3-isopropylmalate dehydratase gene and the branched-chain amino acid transaminase gene above.

In the above-mentioned recombinant bacteria, the 3-isopropylmalate dehydrogenase, 3-isopropylmalate dehydratase and branched-chain amino acid transaminase biotin carboxylase are respectively LeuB, LeuCD and IlvK proteins or have the same function mutants,

In the above-mentioned recombinant bacteria, preferably, the amino acid sequence of the LeuB protein is shown in SEQ ID NO.2, the amino acid sequence of the LeuC protein is shown in SEQ ID NO.3, and the amino acid sequence of the LeuD protein is shown in SEQ ID NO. As shown in NO.4, the amino acid sequence of the IlvK protein is shown in SEQ ID NO.5.

In the above recombinant bacteria, the 2-isopropylmalate synthase is LeuA protein or a mutant having the same function, preferably, the amino acid sequence of the LeuA protein is shown in SEQ ID NO.1.

In the above-mentioned recombinant bacteria, the original strains are wild bacteria capable of producing lipopeptides and their mutagenic strains, mutant strains or genetically engineered strains.

In the above recombinant bacteria, the original strain is Bacillus subtilis, Bacillus cereus or Pseudomonas.

Among the above recombinant bacteria, optionally, the original strain is Bacillus subtilis THY-7.

The Bacillus subtilis THY-7 was deposited on March 11, 2014 in the General Microorganism Center of China Committee for the Collection of Microorganisms, with the registration number CGMCC No.8906, and disclosed in Chinese patent document CN 105400784A.

Among the above recombinant bacteria, optionally, the original strain is Bacillus subtilis THY-7/Pg3-srfA.

The Bacillus subtilis THY-7/Pg3-srfA refers to adding an inducible strong promoter Pg3 to the Bacillus subtilis THY-7, so that the strong promoter Pg3 controls the expression of surfactin synthase srfA. The inducible strong promoter Pg3, Bacillus subtilis THY-7/Pg3-srfA and their specific preparation methods are disclosed in Chinese patent document CN 105400784A.

In the above recombinant bacteria, the LeuA, LeuB, LeuC and LeuD proteins are controlled by strong promoters, and the IlvK protein is controlled by the original constitutive promoter.

The object of the present invention is also to provide the application of the above-mentioned recombinant bacteria in the production of lipopeptides.

In the above application, the lipopeptide biosurfactant is surfactin.

A method for preparing the above-mentioned recombinant bacteria, comprising the steps of:

Amplified 2-isopropylmalate synthase, 3-isopropylmalate dehydrogenase, 3-isopropylmalate dehydratase gene cluster leuABCD and branched-chain amino acid transaminase organisms with their own constitutive promoters Carboxylase IlvK, LeuABCD and IlvK gene sequences were inserted into the shuttle plasmid to construct an expression plasmid;

The expression plasmid was introduced into the original strain to construct the recombinant strain.

Further, the specific method is:

1. Using upstream and downstream primers, carry out polymerase chain reaction with the Bacillus subtilis genome as a template, amplify and obtain the leuABCD gene, preferably, its nucleic acid sequence is shown in SEQ ID NO.6. Similarly, the ilvK gene is amplified and obtained, preferably, its nucleic acid sequence is shown in SEQ ID NO.7.

2. Carry out double digestion of the leuABCD and ilvK genes and the shuttle plasmid respectively, and use ligase to connect the two digested products to obtain the ligated product.

3. The ligation product was transformed into E. coli TOP10 competent cells, and the positive clones were screened for resistance to obtain the expression plasmid pJMP-leuABCD-ilvK containing the gene sequences of leuABCD and ilvK.

4. Transfer the expression plasmid pJMP-leuABCD-ilvK into the starting strain to obtain genetically engineered bacteria transformed with the pJMP-leuABCD-ilvK plasmid.

In the above method for preparing genetically engineered bacteria, the method for constructing the shuttle plasmid pJMP has been disclosed in Chinese patent document CN109097315A.

A method for preparing the above-mentioned recombinant bacteria, optionally, the specific method includes the following:

1. Using leuABCD-F and leuABCD-R as upstream and downstream primers, and using the Bacillus subtilis genome as a template to carry out polymerase chain reaction, amplify and obtain the leuABCD gene sequence, its nucleic acid sequence is shown in SEQ ID NO.6; with ilvK-F and ilvK-R are the upstream and downstream primers, and the polymerase chain reaction is carried out using the Bacillus subtilis genome as a template to amplify and obtain the ilvK gene sequence, and its nucleic acid sequence is shown in SEQ ID NO.7;

2. Digest the leuABCD gene with Xba I and Mlu I, the ilvK gene with Mlu I and Nco I, and the shuttle plasmid with Xba I and Nco I, purify the digested product, and connect with T4 DNA The enzyme connects the three digestion products to obtain a connection product.

3. Transform the ligation product into E. coli TOP10 competent cells, smear it on a kanamycin LB plate, and place it upside down in a 37°C incubator for overnight culture. The resistant clones grown on the plate were picked and cultured, the plasmid was extracted for enzyme digestion and sequencing verification, and the expression plasmid pJMP-leuABCD-ilvK containing the correct leuABCD-ilvK gene sequence was obtained.

4. Transfer the expression plasmid pJMP-leuABCD-ilvK into Bacillus subtilisTHY-7/Pg3-srfA (CN 105400784A) by electroporation, and spread it on the LB plate containing chloramphenicol and kanamycin , picked resistant clones for culture, and carried out PCR verification to obtain genetically engineered bacteria B. subtilis THY-7/Pg3-srfA (leuABCD-ilvK) transformed with pJMP-leuABCD-ilvK plasmid.

Preferably, the Bacillus subtilis genome described in step 1 can be selected from Bacillus subtilis 1012wt (MoBiTec company), Bacillus subtilis THY-7 (CN 105400784A), THY-8 (Chemical Progress, 2013, 32: 2952-2956) , THY-15 (Journal of industrial microbiology & biotechnology. 2015, 42(8): 1139-1147) or other Bacillus strains carrying leuABCD and ilvK genes.

Preferably, the shuttle plasmid described in step 2 is preferably pJMP, and the construction method has been disclosed in Chinese patent document CN109097315A.

The object of the present invention is also to provide the application of the above-mentioned genetically engineered bacteria in the preparation of lipopeptides.

Optionally, using the above-mentioned recombinant bacteria to produce lipopeptides, the steps are as follows:

Insert the recombinant bacteria into the culture medium, carry out expanded culture, and obtain the genetically engineered bacteria liquid;

The engineered bacteria liquid obtained in the step (1) is inserted into the fermentation medium according to 1-20% volume percentage, and the fermentation culture is carried out to obtain a lipopeptide-containing fermentation liquid.

In the above-mentioned method, the method for expanding the cultivation is: culturing for 10-20 hours at a temperature of 35-40° C. and a shaker rotation speed of 150-200 rpm.

In the above method, the method of fermentation culture is to add 0.5-1.5mM IPTG inducer after culturing for 1.5-4h under the condition of 35-40°C and shaker rotation speed of 150-200rpm, and then continue culturing for 40-60h.

In the above method, the composition of the fermentation medium is: sugar 30-100g/L, inorganic nitrogen source 10-50g/L, organic nitrogen source 0.5-3g/L, KH ₂ PO ₄ 0.1-1g/L, Na ₂ HPO ₄ ·12H ₂ O 0.5-0.3g/L,CaCl ₂ 0.002-0.01g/L,MnSO ₄ ·H ₂ O 0.002-0.01g/L,FeSO ₄ ·7H ₂ O 0.002-0.01g/L,pH 6.5-7.5.

Advantages and beneficial effects of the present invention:

The invention uses genetic engineering technology to construct recombinant bacteria, amplifies and expresses 2-isopropylmalate synthase LeuA and 3-isopropylmalate dehydrogenation in the leucine synthesis pathway in lipopeptide-producing Bacillus subtilis cells The enzymes LeuB, 3-isopropylmalate dehydratase LeuCD and branched-chain amino acid transaminase IlvK strengthen the synthesis of leucine in the molecular structure of surfactin, thereby significantly increasing the yield of lipopeptide. Compared with the original strain, the recombinant bacterium obtained by the present invention with overexpressed part of the leucine synthesis pathway has a maximum increase of 55.6% in the production of surfactin, and can be used for the production of lipopeptide biosurfactants, and has good industrial application prospects. The average yield of lipopeptides in the fermentation broth obtained from bottle fermentation is 13-16g/L.

Description of drawings

Figure 1 is a schematic diagram of the plasmid pJMP used for gene overexpression.

Figure 2 is a PCR verification map of the overexpression plasmid pJMP-leuABCD-ilvK containing the leuABCD-ilvK gene string: Swimming lane 1 is the DNA molecular weight standard, and swimming lane 2 is the result of PCR amplification of the plasmid using primers ilvK-F and ilv-R , a 1.3kb band can be obtained.

Fig. 3 is Bacillus subtilis-large coli carrying 2-isopropylmalate synthase LeuA, 3-isopropylmalate dehydrogenase LeuB, 3-isopropylmalate dehydratase LeuCD and branched-chain amino acid transaminase IlvK genes Schematic representation of the Bacillus shuttle plasmid pJMP-leuABCD-ilvK.

Figure 4 shows the genetically engineered bacteria B overexpressing 2-isopropylmalate synthase LeuA, 3-isopropylmalate dehydrogenase LeuB, 3-isopropylmalate dehydratase LeuCD and branched-chain amino acid transaminase IlvK. PCR verification diagram of subtilisTHY-7/Pg3-srfA (leuABCD-ilvK): Lane 1 is the result of PCR amplification of THY-7/Pg3-srfA (pJMP-yngH) with primer ilvK-F and universal primer pJMP-R, A 1.5kb band was obtained. Lane 2 is DNA molecular weight standard.

Figure 5 shows the concentration of surfactin in the fermentation product of the original strain THY-7/Pg3-srfA and the genetically engineered strain THY-7/Pg3-srfA (leuABCD-ilvK).

Detailed ways

The present invention will be further described below in conjunction with the accompanying drawings and specific embodiments. Unless otherwise specified, the biochemical reagents used in the examples are all commercially available reagents, and the technical means used in the examples are conventional means in the books of those skilled in the art.

In the leucine synthesis pathway: glucose generates pyruvate through the glycolysis pathway, and pyruvate is the precursor of leucine synthesis. In the process of producing α-ketoisovaleric acid from pyruvate, the synthesis of acetolactate from pyruvate is the rate-limiting step in the process. The enzyme catalyzing this step is acetohydroxyacid synthase, encoded by the gene ilvHB and the gene alsS. In the process of synthesizing L-leucine from α-ketoisovaleric acid and acetyl CoA, the rate-limiting step is the synthesis of α-isopropylmalate catalyzed by α-isopropylmalate synthase, the gene encoding the enzyme is leuA .

The inventors expressed these three related genes in previous experiments and found that the synthesis of surfactin was significantly reduced when alsS and ilvHB were overexpressed, and the yield of surfactin was increased when leuA was overexpressed, and the lipopeptide yield was 11.58g/L. When leuAB was overexpressed, the production of lipopeptide was not further improved.

Example 1 Construction of plasmids carrying genes leuABCD and ilvK in the leucine synthesis pathway

Pick a single colony of Bacillus subtilis B.subtilis THY-7, inoculate it in LB liquid medium, place it in a shaker at 37°C and 200rpm for overnight culture, and collect the bacteria under the condition of centrifugation at 12000rpm and 5min. Bacterial Genome Extraction Kit to extract THY-7 genome. Using the obtained genome as a template, the upstream primer leuABCD-F (sequence shown in SEQ ID NO.8) and the downstream primer leuABCD-R (sequence shown in SEQ ID NO.9) were used for PCR amplification to obtain a leuABCD fragment. Use upstream primer ilvK-F (sequence shown in SEQ ID NO.10) and downstream primer ilvK-R (sequence shown in SEQ ID NO.11) to carry out PCR amplification to obtain ilvK fragment primers provided by Boshang Biotechnology (Shanghai) Co., Ltd., dissolved in sterile water and diluted to 10 μM for use. The polymerase, buffer and restriction endonuclease used in PCR amplification were purchased from TaKaRa Company. The PCR amplification reaction system is:

The thermal cycling conditions are

The gene fragment of Bacillus subtilis leuABCD was amplified and purified, and its sequence was shown in SEQ ID NO.6. The gene fragment of Bacillus subtilis ilvK was amplified and purified, and its sequence was shown in SEQ ID NO.7. Digest the leuABCD gene with XbaI and MluI, the ilvK gene with MluI and NcoI, and the shuttle plasmid with XbaI and NcoI. Digest at 27-33°C for 1-3 hours and use Omega The company's DNA purification kit was purified, and then ligated overnight at 16-22°C using T4 DNA ligase (NEB Company). The ligation product was transformed into Escherichia coli E. coli TOP10 competent cells (TianGen Company), spread on LB plates containing kanamycin, and cultured overnight in a 37°C incubator upside down. Pick the resistant clones grown on the plate for colony PCR, use the picked colonies as templates, use ilvK-F and plasmid primer ilvK-R as primers, and amplify about 1.3Kb bands (as shown in Figure 2) The positive clones were picked and cultured, the plasmids were extracted and verified by sequencing, and the expression plasmid pJMP-leuABCD-ilvK containing the genes of leuABCD and ilvK was obtained. Figure 1 is a schematic diagram of the plasmid pJMP.

Example 2 Construction of the genetically engineered bacteria B.subtilis THY-7/Pg3-srfA (leuABCD-ilvK) overexpressing the leucine synthesis pathway

The Bacillus subtilis constructed in Example 1 carrying 2-isopropylmalate synthase LeuA, 3-isopropylmalate dehydrogenase LeuB, 3-isopropylmalate dehydratase LeuCD and branched-chain amino acid transaminase IlvK The Bacillus-Escherichia coli shuttle plasmid pJMP-leuABCD-ilvK is used to transform the competent cells of Bacillus subtilis THY-7/Pg3-srfA by electroporation, and the genetically engineered bacteria THY-7/ Pg3-srfA (leuABCD-ilvK). Wherein, the preparation and electrotransformation of Bacillus subtilis THY-7/Pg3-srfA competent cells adopt the method in Chinese patent document CN105400784A.

After resuscitation, take 100uL of bacterial liquid and spread it on LB solid medium containing 10-30μg/mL kanamycin, place it upside down in a 37°C incubator for overnight culture, pick a single colony, and use upstream and downstream primers ilvK-F and pJMP -R for PCR verification, a band of about 1.5kb can be amplified, the verification results are shown in Figure 4, that is, the overexpression of 2-isopropylmalate synthase LeuA and 3-isopropylmalate dehydrogenase LeuB , 3-isopropylmalate dehydratase LeuCD and branched-chain amino acid transaminase IlvK genetically engineered bacteria THY-7/Pg3-srfA (leuABCD-ilvK). The sequence of the plasmid primer pJMP-R is shown in SEQ ID NO.12.

Embodiment 3 uses genetically engineered bacteria THY-7/Pg3-srfA (leuABCD-ilvK) to produce lipopeptide surfactant-surfactin

The genes obtained in Example 2 for overexpressing 2-isopropylmalate synthase LeuA, 3-isopropylmalate dehydrogenase LeuB, 3-isopropylmalate dehydratase LeuCD and branched-chain amino acid transaminase IlvK The engineering bacteria THY-7/Pg3-srfA (leuABCD-ilvK) was inoculated in LB liquid medium (containing chloramphenicol and kanamycin), cultured at 37°C and 200rpm for 16h, and inserted into the container at a ratio of 5%. In a shake flask with 100 mL of fermentation medium, add IPTG when culturing at 37°C and 200 rpm for 2-6 hours, and continue culturing for 2-3 days to obtain a lipopeptide-containing fermentation broth.

The detection of surfactin in the fermentation broth adopts the method of Yu Huimin et al. (Chinese patent CN 105400784A). The statistical results of surfactin concentration in the fermentation broth of the genetically engineered bacteria THY-7/Pg3-srfA (leuABCD-ilvK) and the starting strain THY-7/Pg3-srfA are shown in Figure 5: THY-7/Pg3-srfA (leuABCD -ilvK) Surfactin production can be up to 15.8g/L, which is 54.9% higher than THY-7/Pg3-srfA starting bacteria (10.2g/L). When THY-7/Pg3-srfA(leuABCD-ilvK) was cultured in a 5L fermenter, the highest yield of surfactin was 19g/L.

The above embodiments are only used to illustrate the technical solutions of the present invention, and are not intended to limit them; although the present invention has been described in detail with reference to the foregoing embodiments, those of ordinary skill in the art should understand that: it can still be applied to the foregoing embodiments The technical solutions described in the examples are modified, or some or all of the technical features are equivalently replaced; and these modifications or replacements do not make the essence of the corresponding technical solutions depart from the scope of the technical solutions of the embodiments of the present invention.

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465 470

<210> 4

<211> 199

<212> PRT

<213> Bacillus subtilis

<400> 4

Met Glu Pro Leu Lys Ser His Thr Gly Lys Ala Ala Val Leu Asn Arg

1 5 10 15

Ile Asn Val Asp Thr Asp Gln Ile Ile Pro Lys Gln Phe Leu Lys Arg

20 25 30

Ile Glu Arg Thr Gly Tyr Gly Arg Phe Ala Phe Phe Asp Trp Arg Tyr

35 40 45

Asp Ala Asn Gly Glu Pro Asn Pro Glu Phe Glu Leu Asn Gln Pro Val

50 55 60

Tyr Gln Gly Ala Ser Ile Leu Ile Ala Gly Glu Asn Phe Gly Cys Gly

65 70 75 80

Ser Ser Arg Glu His Ala Pro Trp Ala Leu Asp Asp Tyr Gly Phe Lys

85 90 95

Ile Ile Ile Ala Pro Ser Phe Ala Asp Ile Phe His Gln Asn Cys Phe

100 105 110

Lys Asn Gly Met Leu Pro Ile Arg Met Pro Tyr Asp Asn Trp Lys Gln

115 120 125

Leu Val Gly Gln Tyr Glu Asn Lys Ser Leu Gln Met Thr Val Asp Leu

130 135 140

Glu Asn Gln Leu Ile His Asp Ser Glu Gly Asn Gln Ile Ser Phe Glu

145 150 155 160

Val Asp Pro His Trp Lys Glu Met Leu Ile Asn Gly Tyr Asp Glu Ile

165 170 175

Ser Leu Thr Leu Leu Leu Glu Asp Glu Ile Lys His Phe Glu Ser Gln

180 185 190

Arg Ser Ser Trp Leu Gln Ala

195

<210> 5

<211> 363

<212> PRT

<213> Bacillus subtilis

<400> 5

Met Thr Lys Gln Thr Ile Arg Val Glu Leu Thr Ser Thr Lys Lys Pro

1 5 10 15

Lys Pro Asp Pro Asn Gln Leu Ser Phe Gly Arg Val Phe Thr Asp His

20 25 30

Met Phe Val Met Asp Tyr Ala Ala Asp Lys Gly Trp Tyr Asp Pro Arg

35 40 45

Ile Ile Pro Tyr Gln Pro Leu Ser Met Asp Pro Ala Ala Met Val Tyr

50 55 60

His Tyr Gly Gln Thr Val Phe Glu Gly Leu Lys Ala Tyr Val Ser Glu

65 70 75 80

Asp Asp His Val Leu Leu Phe Arg Pro Glu Lys Asn Met Glu Arg Leu

85 90 95

Asn Gln Ser Asn Asp Arg Leu Cys Ile Pro Gln Ile Asp Glu Glu Gln

100 105 110

Val Leu Glu Gly Leu Lys Gln Leu Val Ala Ile Asp Lys Asp Trp Ile

115 120 125

Pro Asn Ala Glu Gly Thr Ser Leu Tyr Ile Arg Pro Phe Ile Ile Ala

130 135 140

Thr Glu Pro Phe Leu Gly Val Ala Ala Ser His Thr Tyr Lys Leu Leu

145 150 155 160

Ile Ile Leu Ser Pro Val Gly Ser Tyr Tyr Lys Glu Gly Ile Lys Pro

165 170 175

Val Lys Ile Ala Val Glu Ser Glu Phe Val Arg Ala Val Lys Gly Gly

180 185 190

Thr Gly Asn Ala Lys Thr Ala Gly Asn Tyr Ala Ser Ser Leu Lys Ala

195 200 205

Gln Gln Val Ala Glu Glu Lys Gly Phe Ser Gln Val Leu Trp Leu Asp

210 215 220

Gly Ile Glu Lys Lys Tyr Ile Glu Glu Val Gly Ser Met Asn Ile Phe

225 230 235 240

Phe Lys Ile Asn Gly Glu Ile Val Thr Pro Met Leu Asn Gly Ser Ile

245 250 255

Leu Glu Gly Ile Thr Arg Asn Ser Val Ile Ala Leu Leu Lys His Trp

260 265 270

Gly Leu Gln Val Ser Glu Arg Lys Ile Ala Ile Asp Glu Val Ile Gln

275 280 285

Ala His Lys Asp Gly Ile Leu Glu Glu Ala Phe Gly Thr Gly Thr Ala

290 295 300

Ala Val Ile Ser Pro Val Gly Glu Leu Ile Trp Gln Asp Glu Thr Leu

305 310 315 320

Ser Ile Asn Asn Gly Glu Thr Gly Glu Ile Ala Lys Lys Leu Tyr Asp

325 330 335

Thr Ile Thr Gly Ile Gln Lys Gly Ala Val Ala Asp Glu Phe Gly Trp

340 345 350

Thr Thr Glu Val Ala Ala Leu Thr Glu Ser Lys

355 360

<210> 6

<211> 4755

<212>DNA

<213> Bacillus subtilis

<400> 6

atgcgcaaaa ttaatttttt cgatacgacg cttcgtgatg gtgaacagtc ccctggagtg 60

aacttgaata cacaggagaa acttgccata gctaagcagc tcgaaagact cggggcagat 120

atcattgaag cgggatttcc cgcttcgtcc cgaggtgact ttttagctgt tcaggaaatc 180

gcaagaacca ttaaaaattg ttcagtaact ggtctggccc gttgtgtaaa aggtgatatt 240

gatgctgctt gggaagcgtt aaaagaaggt tctcacccga gaattcatgt ttttatcgcc 300

acatcggaca ttcatttgaa gcacaagctg aaaatgacac gtgagcaagt cattgaaaga 360

gcggttgaaa tggtgaaata cgcaaaagaa cgttttccga ttgtgcaatg gtcagctgaa 420

gatgcctgcc gcactgaact gccgtttcta gcagaaatcg tcgaaaaagt gattgacgca 480

ggcgccagtg ttatcaatct tccggacact gtcggctacc tggctccggc ggaatacgga 540

aatatcttta gatatatgaa ggaaaacgtt ccgaacattc acaaagcaaa gctttcagcc 600

cactgtcatg atgatttagg aatggcagtc gcaaactctc ttgctgcgat tgaaaatggc 660

gctgatcaaa tcgaatgcgc tgtgaacggg atcggtgaaa gagccggaaa cgcggcatta 720

gaggaaattg ccgtagccct ccataccaga aaagatttct accaagtcga aacaggcatt 780

acactgaacg agattaagag aacaagtgat ttagtaagca aactgacagg catggctgtc 840

ccgcgcaaca aagcggttgt tggagataat gcatttgctc atgaatcagg catccatcag 900

gacggctttt taaaggaaaa atcgacttat gaaattattt caccggagct tgtcggcgta 960

accgcagatg cgcttgtcct aggtaaacat tccggacgcc acgcatttaa agaccggctg 1020

actgctttag gattccaatt tgacagtgaa gagattaata aattctttac gatgttcaaa 1080

gagttgactg agaagaaaaa agaaatcact gatgaggatc ttgtttctct tattttagaa 1140

gaaaaagtaa cagatcgcaa gattgggtat gaatttcttt ctctgcaagt aattacgga 1200

acaagtcagg tccctacggc tactctttcg ttgaaaaatc aagaaaacgc agagcttatt 1260

caggaagctg caactggagc tggaagtgtg gaagcaatct acaatacgct tgagcgctgc 1320

atcgataagg acgtggagct cttagactac cgcattcagt ctaacagaaa aggcgaagat 1380

gcatttgccc aggtgtatgt aagagttttg gtgaacggaa aagaatcagc aggtcggggc 1440

atagcgcaag acgtattaga agcatcagcg aaagcctatt tgaacgcagt caaccgtcaa 1500

ttggttttcc agtcgaatat gagcggattg aaaaaccaca cagctgtcgg atcataaaag 1560

aaaggagaac ggttaacttg aagaaacgta ttgctctatt gcccggagaac gggatcggcc 1620

ctgaagtatt agaatcagcg acagacgtac tgaaaagtgt tgccgaacgc tttaaccatg 1680

aatttgaatt tgaatatggc ctgattggag gggcggctat tgatgaacat cataaccccc 1740

tcccggagaa aaccgttgct gcttgtaaaa atgcagacgc gatattgctt ggcgctgtcg 1800

gcggaccgaa atgggatcaa aatccttcgg aactgagacc ggaaaaaggg ctgctcagca 1860

tcagaaaaca gcttgatttg tttgcgaatt tacggcctgt gaaggtattt gaaagccttt 1920

ctgacgcttc gcctttgaaa aaagaatata tagataatgt tgatttcgtt atcgttcgtg 1980

aactcacagg cggcttgtat ttcggccagc cgagcaaacg ctatgtaaac actgaaggtg 2040

agcaggaagc agtagataca ctgttttata agcgaacgga aattgaacga gtgatcagag 2100

agggcttcaa aatggcggca gccagaaaag gcaaagtgac ctctgtagat aaagcgaatg 2160

ttcttgaatc aagccggctg tggcgtgaag tggctgagga cgttgcgaaa gaatttcctg 2220

atgtgaagct tgagcacatg cttgtggata acgcggccat gcagctaatc tatgcaccga 2280

atcaatttga tgtcgttgtg actgaaaata tgttcggtga tattttaagc gatgaagcgt 2340

ccatgcttac aggctcgctc ggaatgctcc cgtcagccag cctatcaagc tctggtcttc 2400

atctgtttga acctgttcat ggctccgcgc ctgatattgc tggtaaaggg atggcaaatc 2460

cgttcgcagc catattgtca gcggcaatgc ttttgaggac atctttcggg cttgaagagg 2520

aagcgaaagc tgtagaagat gcggtaaaca aagtcttggc ttccggaaaa agaacaaaag 2580

acttggcacg gggtgaagag ttcagcagca ctcaggccat tacagaggaa gttaaggcag 2640

caatcatgag tgaaaataca atgtctaatg tgtgacagct tacgttaagc ggtcttagct 2700

ctaggtagag ggaggaaata aaagatgatg cctcgaacaa tcatcgaaaa gatttgggat 2760

cagcatattg taaaacatgg tgagggaaag ccggatcttc tctatattga tttgcacctc 2820

attcatgagg tgacgtctcc tcaggcattt gaaggcttga gacaaaaggg aagaaaggtc 2880

agaagacccc aaaacacatt tgcgacaatg gaccacaaca tcccgactgt caatcgtttt 2940

gagataaagg atgaagttgc gaaacgccag gtaacggcgc ttgaaagaaa ctgtgaggaa 3000

tttggcgtgc gccttgccga tcttcacagc gtggatcaag ggattgtcca tgtcgtcggc 3060

cctgaactgg gcttaacgct tccagggaaa acgattgtgt gcggggacag tcatacatca 3120

acacatggcg ctttcggcgc tcttgcattt ggaatcggga cgagtgaagt cgaacatgtt 3180

ctttccacac agacactttg gcagcagcgt ccaaaaacac ttgaagtgcg cgtagatgga 3240

acgcttcaaa aaggggtaac ggcaaaggat gtcatccttg ctgtcatcgg caaatacggt 3300

gtgaaattcg gcacaggcta cgtcattgaa tacactgggg aagtattcag aaatatgacg 3360

atggatgaac gaatgactgt ttgtaacatg tcaattgaag caggagcaag agcaggtttg 3420

attgcacctg acgaggtgac gtttgaatat tgcaaaaatc gcaagtacac gccaaaaggc 3480

gaagaatttg acaaggccgt agaggaatgg aaggcgctgc gcacagacccc gggcgctgtt 3540

tacgataaat ctatcgtcct tgacggcaac aaaatttccc ctatggtgac atggggcatt 3600

aacccgggaa tggttcttcc tgtcgattct gaagttcctg cgccggaaag cttttctgca 3660

gaagatgata aaaaagaagc gattcgcgct tatgaatata tgggactgac tcctcatcag 3720

aaaattgaag acattaaagt ggagcacgta tttatcggtt cctgcacaaa ttcccgcatg 3780

actgaccttc gccaggctgc tgacatgatc aaggggaaga aggtagctga cagcgtaagg 3840

gccatcgtcg tgcccggatc ccaaagagtg aagcttcagg ctgaaaaaga agggcttgac 3900

caaattttct tggaagctgg atttgaatgg agagagtcag gctgcagcat gtgtttgagt 3960

atgaataatg atgttgttcc tgagggagag cgctgtgcat caacctctaa ccgcaacttc 4020

gagggcagac aaggaaaagg tgcaagaaca catctcgtca gcccggcaat ggctgcgatg 4080

gctgccattc acggacactt cgttgatgtc agaaagtttt atcaggaaaa aacagttgtg 4140

taaggagtgc gcgagatgga acctttgaaa tcacatacgg ggaaagcagc cgtattaaat 4200

cggatcaatg tggatacaga ccagattatt cctaagcaat ttttgaagag gattgaaaga 4260

acaggctacg ggcgttttgc attctttgac tggagatatg atgcgaatgg tgaaccgaac 4320

cctgaatttg aattaaacca gcctgtttat caaggagctt ccattttaat agcaggagaa 4380

aacttcggct gcgggtcatc gcgtgaacat gctccgtggg cacttgatga ttatgggttt 4440

aaaattatca ttgcgccgtc attcgctgat attttccatc agaactgctt taaaaacggc 4500

atgcttccga tccgcatgcc atatgacaat tggaaacagc ttgtcggcca gtatgaaaac 4560

aagtcattgc aaatgactgt tgaccttgaa aatcagctga ttcatgacag tgaaggcaat 4620

caaatttcat ttgaagttga cccgcattgg aaagagatgc tgatcaacgg atatgatgaa 4680

atttcattaa cgctgctgct ggaagatgaa atcaagcatt ttgaatcaca aagaagctct 4740

tggcttcaag cctga 4755

<210> 7

<211> 1247

<212>DNA

<213> Bacillus subtilis

<400> 7

agcgcttata cgcgttttct cctgcttttt tcatatgaat ttcttacaaa tttgagcaaa 60

cctattgcga ttatttgttg aaggtataca atagaatata attattttca aataagtttg 120

ataatataaa caatttaaca gcagggagat tgaccatgac taaacaaaca attcgcgttg 180

aattgacatc aacaaaaaaa ccgaaaccag acccaaatca gctttcgttc ggaagagtgt 240

ttacagacca catgtttgta atggactatg ccgcagataa aggttggtac gatccaagaa 300

tcattcctta tcagccctta tcaatggatc cggccgcaat ggtctatcac tacggccaaa 360

ccgtgtttga agggttaaag gcttacgtgt cagaggatga ccatgttctg cttttcagac 420

cggaaaaaaa tatggaacgc ctgaatcaat caaacgaccg cctctgcatc ccgcaaattg 480

atgaagaaca ggttcttgaa ggcttaaagc agcttgtcgc aattgataaa gactggattc 540

caaatgcgga gggcacgtcc ctatacatcc gtccgttcat catcgcaacc gagcctttcc 600

ttggtgttgc ggcatctcat acgtataagc tcttgatcat tctttctccg gtcggctctt 660

attacaaaga aggcattaag ccggtcaaaa tcgctgttga aagtgaattt gtccgtgcgg 720

taaaaggcgg aacaggaaat gccaaaaccg cagggaacta cgcttcaagc ttaaaagcgc 780

agcaggtcgc cgaagagaaa ggattttccc aagtgctttg gctggacggc attgagaaga 840

aatacatcga agaagttgga agcatgaaca tcttcttcaa aatcaacggt gaaatcgtaa 900

cgccgatgct gaacggaagc atcctggaag gcattacgcg caattcagtc atcgccttgc 960

ttaagcattg gggccttcaa gtttcagaac ggaaaattgc gatcgatgag gtcatccaag 1020

cccataaaga cggcatcctg gaagaagcct tcggaacagg tacagcagct gttatttccc 1080

cagtcggcga gctgatctgg caggatgaaa cactttcgat caacaacggt gaaacaggag 1140

aaatcgcaaa aaaactatat gacacgatta caggcattca aaaaggcgct gtcgcagacg 1200

aattcggatg gacgaccgaa gttgcagcgc tgactgaaag caagtaa 1247

<210> 8

<211> 35

<212>DNA

<213> Artificial Sequence

<400> 8

cactctagaa tgcgcaaaat taattttttc gatac 35

<210> 9

<211> 35

<212>DNA

<213> Artificial Sequence

<400> 9

taacgcgttc aggcttgaag ccaagagctt ctttg 35

<210> 10

<211> 34

<212>DNA

<213> Artificial Sequence

<400> 10

acaacgcgtt ttctcctgct tttttcatat gaat 34

<210> 11

<211> 29

<212>DNA

<213> Artificial Sequence

<400> 11

cacccatggt tacttgcttt cagtcagcg 29

<210> 12

<211> 20

<212>DNA

<213> Artificial Sequence

<400> 12

cagcctttgttggttctttt 20

Claims (10)

1. a kind of recombinant bacterium of high yield lipopeptid is by the 2-Isopropylmalate synthase genetic transformation in leucine route of synthesis It is built-up to original strain.

2. recombinant bacterium according to claim 1, which is characterized in that it is de- that the recombinant bacterium has also been transferred to 3- isopropylmolic acid One or more in hydrogenase gene, 3-Isopropylmalate dehydratase gene and branched-chain amino acid aminotransferase gene.

3. recombinant bacterium according to claim 2, which is characterized in that the 3-Isopropylmalate dehydrogenase, 3- isopropyl Malic acid dehydratase and branched-chain amino acid transaminase biotin carboxylase is respectively LeuB, LeuCD and IlvK albumen or it has The mutant of identical function, it is preferred that the amino acid sequence of the LeuB albumen is as shown in SEQ ID NO.2, the LeuC egg White amino acid sequence is as shown in SEQ ID NO.3, and the amino acid sequence of the LeuD albumen is as shown in SEQ ID NO.4, institute The amino acid sequence of IlvK albumen is stated as shown in SEQ ID NO.5.

4. recombinant bacterium according to claim 1, which is characterized in that the 2-Isopropylmalate synthase be LeuA albumen or Its mutant with the same function, it is preferred that the amino acid sequence of the LeuA albumen is as shown in SEQ ID NO.1.

5. recombinant bacterium according to claim 1-4, which is characterized in that the original strain is with production lipopeptid energy Strain is transformed in the wild mushroom and its mutagenic fungi of power, mutant strain or genetic engineering.

6. recombinant bacterium according to claim 5, which is characterized in that the original strain is bacillus subtilis, wax-like bud Spore bacillus or pseudomonad.

7. recombinant bacterium according to claim 6, which is characterized in that the original strain is bacillus subtilis Bacillus subtilis THY-7。

8. recombinant bacterium according to claim 7, which is characterized in that the original strain is bacillus subtilis Bacillus subtilis THY-7/Pg3-srfA。

9. recombinant bacterium according to claim 8, which is characterized in that LeuA, LeuB, LeuC and LeuD albumen is opened by force Mover control, the IlvK albumen are controlled by former constitutive promoter.

10. application of the described in any item recombinant bacteriums of claim 1-9 in production lipopeptid.